Post-Translational Modifications on HLA Class I Presented Peptides

Caron Laboratory


Study Overview

This analysis characterizes post-translational modifications (PTMs) identified on HLA class I peptides from the JY cell line using data-dependent acquisition mass spectrometry. All analyses are restricted to peptides of length 8-14 amino acids.

HLA Alleles: A\*02:01, B\*07:02, C\*07:02


Data Summary

DatasetPeptides
Background (unmodified)77,100
**Total Modified****8,804**
Cysteinylation2,355
Deamidation2,081
Oxidation1,640
Phosphorylation702
Acetylation510
Methylation475
Ubiquitination426
Dimethylation319
Citrullination250
N-Glycosylation42
SUMOylation4

PTM Categories

PTM TypeTarget Residues
PhosphorylationS, T, Y
AcetylationK, N-terminus
CysteinylationC
MethylationK, R
DimethylationK, R
DeamidationN, Q
Biological OxidationP, I, L, Q, S, T, V, C, D, E, N, Y, G, K, R
CitrullinationR
UbiquitinationK
N-GlycosylationN
SUMOylationK

HLA Binding Classification

CategoryEL Rank Threshold
Strong binder< 0.5%
Weak binder0.5% – 2%
Non-binder≥ 2%

Figure 1: PTM Composition

Figure 1A: PTM Distribution

![Figure 1A](figure_panels/Figure1A_BarChart.png)
![Figure 1A](figure_panels/Figure1A_BarChart.png)

Distribution of unique modified peptides by PTM type (length 8-14 amino acids).

Figure 1B: PTM Proportions

![Figure 1B](figure_panels/Figure1B_Donut.png)
![Figure 1B](figure_panels/Figure1B_Donut.png)

Proportional representation of each PTM class within the modified peptidome.


Figure 2: Peptide Length Distribution

![Figure 2](figure_panels/Figure2_Length_Distribution.png)
![Figure 2](figure_panels/Figure2_Length_Distribution.png)

Ridgeline density plot comparing peptide length distributions across PTM types. Gray distribution represents unmodified background peptides.


Figure 4A: PTM Landscape

![Figure 4A](figure_panels/Figure4A_Circos_PTM_Landscape.png)
![Figure 4A](figure_panels/Figure4A_Circos_PTM_Landscape.png)

Circos diagram illustrating relationships between PTM types (outer sectors), modified residues (links), and HLA binding alleles (inner elements). Link density reflects relative abundance of each PTM-residue combination.


Figure 4B: Positional Enrichment

By PTM and Residue

![Figure 4B](figure_panels/Figure4B_Position_Enrichment.png)
![Figure 4B](figure_panels/Figure4B_Position_Enrichment.png)

Log₂ enrichment of PTM occurrence at peptide positions 8-14 relative to background amino acid positional distribution. PTM+residue combinations with ≥20 occurrences. Color scale: blue (depletion) to red (enrichment), capped at ±3. Correlation column indicates similarity to background distribution.

By PTM Type (Aggregated)

![Figure 4B2](figure_panels/Figure4B2_Position_Enrichment_ByPTM.png)
![Figure 4B2](figure_panels/Figure4B2_Position_Enrichment_ByPTM.png)

Aggregated positional enrichment by PTM type (≥50 occurrences), combining all modified residues.


Figure 4C: PTM Position Density

![Figure 4C](figure_panels/Figure4C_Position_Density.png)
![Figure 4C](figure_panels/Figure4C_Position_Density.png)

Density curves showing modification site distribution across peptide positions 8-14. PTM+residue combinations with ≥50 modifications.


Figure 5A: HLA Binding Affinity

Strong Binders by PTM and Residue

![Figure 5A Strong by Residue](figure_panels/Figure5A_StrongBinders_ByResidue.png)
![Figure 5A Strong by Residue](figure_panels/Figure5A_StrongBinders_ByResidue.png)

Percentage of strong HLA binders (EL Rank < 0.5%) for each PTM+residue combination across alleles A0201, B0702, and C0702. Combinations with ≥10 peptides.

Weak Binders by PTM and Residue

![Figure 5A Weak by Residue](figure_panels/Figure5A_WeakBinders_ByResidue.png)
![Figure 5A Weak by Residue](figure_panels/Figure5A_WeakBinders_ByResidue.png)

Percentage of weak HLA binders (0.5% ≤ EL Rank < 2%) for each PTM+residue combination.

Strong Binders by PTM Type

![Figure 5A Strong by PTM](figure_panels/Figure5A_StrongBinders_ByPTM.png)
![Figure 5A Strong by PTM](figure_panels/Figure5A_StrongBinders_ByPTM.png)

Strong binder percentages aggregated by PTM type across alleles.

Weak Binders by PTM Type

![Figure 5A Weak by PTM](figure_panels/Figure5A_WeakBinders_ByPTM.png)
![Figure 5A Weak by PTM](figure_panels/Figure5A_WeakBinders_ByPTM.png)

Weak binder percentages aggregated by PTM type.


Figure 5B: EL Rank Distribution

![Figure 5B](figure_panels/Figure5B_ELRank_Distribution.png)
![Figure 5B](figure_panels/Figure5B_ELRank_Distribution.png)

EL Rank distribution comparison between modified peptides (colored outlines) and background unmodified peptides (gray) within the binder range (EL Rank 0-2). Dashed lines indicate strong binder (0.5%) and weak binder (2%) thresholds.


Figure 6: Specific PTM Analyses

Figure 6A: Doubly Phosphorylated Peptides

![Figure 6A](figure_panels/Figure6A_DoublyPhospho_Sites.png)
![Figure 6A](figure_panels/Figure6A_DoublyPhospho_Sites.png)

Site distribution for peptides carrying two or more phosphorylation sites.

Figure 6B: Ubiquitination Remnants

![Figure 6B](figure_panels/Figure6B_Ubiq_G_vs_GG.png)
![Figure 6B](figure_panels/Figure6B_Ubiq_G_vs_GG.png)

Comparison of ubiquitin remnant types following tryptic digestion: single glycine (G, +57 Da) versus diglycine (GG, +114 Da).

Figure 6C: Oxidation Classification

![Figure 6C](figure_panels/Figure6C_Oxidation_Bio_vs_Art.png)
![Figure 6C](figure_panels/Figure6C_Oxidation_Bio_vs_Art.png)

Classification of oxidized peptides: biological oxidation (P, I, L, Q, S, T, V, C, D, E, N, Y, G, K, R) versus artifact oxidation (M, W, H).

Figure 6D: Biological Oxidation by Residue

![Figure 6D](figure_panels/Figure6D_BioOxid_BindingByResidue.png)
![Figure 6D](figure_panels/Figure6D_BioOxid_BindingByResidue.png)

HLA binding classification for biologically oxidized peptides stratified by oxidized residue. Sorted by percentage of strong binders.

Figure 6D2: Biological Oxidation Heatmap

![Figure 6D2](figure_panels/Figure6D2_BioOxid_Heatmap.png)
![Figure 6D2](figure_panels/Figure6D2_BioOxid_Heatmap.png)

Strong binder percentages for biological oxidation cross-tabulated by residue and HLA allele.

Figure 6E: Glycan Complexity

![Figure 6E](figure_panels/Figure6E_Glycan_Complexity.png)
![Figure 6E](figure_panels/Figure6E_Glycan_Complexity.png)

N-glycosylation analysis stratified by glycan complexity (sum of monosaccharide units). Categories: Simple (1-3), Medium (4-8), Complex (9-15).

Figure 6E2: Glycan Types

![Figure 6E2](figure_panels/Figure6E2_Glycan_Types_Detail.png)
![Figure 6E2](figure_panels/Figure6E2_Glycan_Types_Detail.png)

Individual glycan structures with HLA binding classification, sorted by monosaccharide count.


Analysis Date: January 2026 | Made by: Peter Kubiniok